The invention relates generally to chromosome inheritance and cell proliferative disorders and more specifically to a polypeptide and a polynucleotide that encodes a polypeptide that affects organization of chromosomes within the nucleus and methods of use therefor.
Eukaryotic organisms package DNA via nucleosomes into highly organized and dynamic chromosomes. During the mitotic cell division cycle the chromosomes must first be completely replicated after which the newly formed sister chromatids condense and are segregated equally to the daughter nuclei. After cytokinesis the whole chromosome cycle is ready to be reinitiated. Pre-meiotic replication results in two sets of sister chromatids. During the first meiotic division it is the paired homologs (two sets of sister chromatids) which segregate from each other, then the sister chromatids are separated into four daughter nuclei of the gametes (spores in yeast). These events are highly conserved and occur in all eukaryotes from unicellular yeast to humans.
There are three cis acting elements which are essential to the chromosome cycle in any eukaryote: replication origins, telomeres, and centromeres. Replication is initiated during S-phase from the replication origins. These regions of the chromosome ensure that the chromosomes are completely replicated before the cell embarks on mitosis. Telomeres are specialized structures which allow complete replication of the ends of chromosomes and protect them from erosion and fusion with other DNA fragments. A centromere is a cis-acting DNA element where protein factors required for mitotic and meiotic chromosome transmission (collectively termed the kinetochore) are assembled. Centromeres act as the nucleation point for kinetochore formation and attachment of the sister chromatids to microtubules emanating from each pole of the spindle. The centromeres, in conjunction with normal spindle formation, are responsible for the delivery of a complete set of chromosomes to the daughter nuclei. The kinetochore mediates attachment of the chromosomes to spindle microtubules during mitosis and ensures that each daughter cell receives a complete set of chromosomes. Centromere malfunction results in the loss of or gain of chromosomes, i.e., aneuploidy, which is associated with 45% of spontaneous abortions in humans (Jacobs and Hassold (1995), Ads. Genet. 33, 101-132). In addition, aneuploidy is commonly observed in human cancer (Duesberg et al. Anticancer Res 1999 November-December; 19(6A):4887-906; Sen S., Curr Opin Oncol 2000 January; 12(1):82-8).
Heterochromatin is an enigmatic component of higher eukaryotic genomes. The paucity of genes and abundance of repetitive sequence in heterochromatin contribute to it being described as functionally inert. However, heterochromatin houses essential single copy loci (Wakimoto, B. T., and Hearn, M. G. (1990), Genetics 125, 141-54) and the rDNA loci, the most highly transcribed genes in the genome (reviewed in Williams, S. M., and Robbins, L. G. (1992), Trends Genet 8, 335-40). In addition, the centromere, the site of kinetochore formation, spindle attachment and checkpoint control during mitosis and meiosis is usually buried deep within heterochromatin. Elegant studies from a variety of organisms (Bass et al. (1997), J Cell Biol 137, 5-18; Goday, C., and Pimpinelli, S. (1989), Chromosoma 98, 160-6) indicate that heterochromatin plays other important roles in chromosome inheritance. For example, heterochromatic homology is required for faithful homolog pairing and chromosome segregation during male and female meiosis in Drosophila (Dernburg et al., (1996), Cell 85, 745-59; Karpen et al. (1996), Science 273, 118-22; McKee, B. D., and Karpen, G. H. (1990), Cell 61, 61-72). While these analyses have highlighted essential roles for heterochromatin in inheritance functions, more detailed dissections will entail characterizing the specific gene products that control heterochromatin metabolism and chromosome inheritance.
The conserved heterochromatic location of higher eukaryotic centromeres suggests that intrinsic properties of heterochromatin are important for chromosome inheritance. Based on this hypothesis, mutations in Drosophila melanogaster that alter heterochromatin-induced gene silencing were tested for effects on chromosome inheritance. The invention provides cloning and characterization of the Su(var)2-10 locus, initially identified as a Suppressor of Position Effect Varegation. Su(var)2-10 is required for viability, and mutations cause both minichromosome and endogenous chromosome inheritance defects. These defects include the hypocondensation of mitotic chromosomes and disruptions in the nuclear organization of interphase chromosomes. The Su(var)2-10 locus encodes a member of the PIAS/ARIP3/Miz1 protein family, a highly conserved group with diverse functions. Accordingly, the invention encompasses these related members of the family and methods of using these related members. Despite multiple chromosomal phenotypes SU(VAR)2-10 protein is not found in mitotic chromosomes. Instead, the protein colocalizes with lamin at the nuclear membrane and surrounds or sheaths interphase polytene chromosomes. Su(var)2-10 provides a mechanistic link between nuclear organization, gene expression and chromosome inheritance.
Su(var)2-10 mutants exhibit multiple phenotypes, including dominant alterations in minichromosome inheritance and heterochromatin-induced gene silencing, and recessive lethality, endogenous chromosome inheritance defects and melanotic tumors.
In one embodiment, the invention provides an isolated polypeptide characterized as modulating heterochromatin-induced gene silencing; and which is required for chromosome inheritance.
In another embodiment, the invention provides an isolated polynucleotide encoding a polypeptide characterized as modulating heterochromatin-induced gene silencing; and which is required for chromosome inheritance. In one embodiment, the polypeptide has a sequence as set forth in SEQ ID NO:2 or 4.
In yet another embodiment, the invention provides a transgenic organism having a transgene disrupting expression of a polynucleotide sequence encoding encoding a
Su(var)2-10 polypeptide, or conservative variations thereof, wherein the polynucleotide is chromosomally integrated into the germ cells of the organism.
In yet another embodiment, the invention provides a nucleic acid construct comprising a disrupted polynucleotide sequence encoding SEQ ID NO:2 or 4, wherein the polynucleotide sequence is disrupted by integration of nucleic acid sequence which inhibits expression of a functional gene product.
In another embodiment, the invention provides a method of producing a transgenic organism, comprising: introducing in the genome of the organism a nucleic acid construct comprising a disrupted polynucleotide sequence encoding SEQ ID NO:2 or 4, wherein the polynucleotide sequence is disrupted by integration of nucleic acid sequence which inhibits expression of a functional gene product operably linked to a promoter which functions in the organism to cause the production of an RNA sequence; and obtaining a transgenic organism having a disrupted polynucleotide sequence encoding wild type Su(var)2-10.
In yet another embodiment, the invention provides a transgenic organism encoding the polypeptide sequence of SEQ ID Nos.2 or 4, operably linked to a conditional promoter, chromosomally integrated into the germ cells of the insect.
In another embodiment, the invention provides an insecticide composition, comprising a nucleic acid construct comprising a disrupted polynucleotide sequence encoding SEQ ID NO:2 or 4, wherein the polynucleotide sequence is disrupted by integration of nucleic acid sequence which inhibits expression of a functional gene product and an agriculturally acceptable carrier.
In another embodiment, the invention provides an antibody which interacts with a Su(var)2-10 polypeptide or binds to an antigenic fragment of a Su(var)2-10 polypeptide.
In yet another embodiment, the invention provides a method of identifying an agent which modulates a Su(var)2-10 polypeptide activity, by incubating the agent and the polypeptide or a recombinant cell expressing the polypeptide, under conditions sufficient to allow the components to interact; and determining the effect of the agent on the activity of the polypeptide or the polypeptide""s expression compared to a control.
In yet another embodiment, the invention provides a method of detecting or diagnosing a cell proliferative disorder in a sample, comprising contacting the sample with an agent which detects a mutant Su(var)2-10, wherein the presence of the mutant Su(var)2-10 is indicative of a cell proliferative disorder.